This invention relates to a vaccine for poultry to prevent salmonellosis and other microorganism-related health problems in humans. In particular, this invention relates to use of a heterophil-adapted strain of microorganism to vaccinate poultry.
Salmonellosis is caused by a group of gram negative bacteria from the genus Salmonella, and is responsible for approximately 2 million cases of food poisoning annually in the United States. Salmonellosis caused by Salmonella enteritidis (SE) in eggs has been the most important food-born public health Salmonella hazard.
Most outbreaks have been traced to consumption of insufficiently cooked eggs. A number of phage types of SE exist but the majority of outbreaks have been traceable to a single or a few phage types of SE. Human SE food poisoning epidemics have also been reported from many other countries, but the phage types reported have not necessarily been those prevailing in the United States.
Some avian strains of SE are vertically transmitted to the eggs of laying hens. The ovaries, oviduct and isthmus have been identified as sites of vertical transmission. In addition, some observations have pointed to cloacal infection of the egg. It is not known which strains or phage types of SE are vertically transmitted, and genetic or molecular requirements for vertical transmission are not known. Infection of hens with SE has led to colonization of the ceca and of the reproductive organs, usually without disease. In most instances infected hens have continued to lay eggs at normal frequencies. Because of absence of overt disease, infection has been difficult to detect clinically or by serologic diagnostic procedures.
Detection of SE in the hen house, individual hens and/or eggs is a difficult task, with a high degree of uncertainty. In the absence of total eradication of infection, vaccination remains the method of choice for disease control. Because SE does not usually cause disease in hens, there is not a vigorous immune response, and hens remain carriers for long periods, even for life. Therefore, it is presumed that immunization will have to be repeatedly administered. Only live attenuated vaccines given orally can be expected to be efficacious in salmonellosis. Curtiss, R. III, et al., Vet. Microbiol. 37: 397-405 (1993). It is possible that a vaccine strain may have to displace the egg-transmitted strain in the gut and possibly the reproductive tract of hens.
Salmonellosis due to SE is in most instances not a poultry disease but one of the most serious public health hazards worldwide. Prevention of the risk of SE transmission from ingested eggs would save several thousands of lives and would save around $1 billion annually. Thus, a vaccine for poultry against transmission of Salmonella would reduce occurrence of salmonellosis in humans and make a significant contribution to public health worldwide.
The present invention features a method of producing a heterophil-adapted strain of microorganism suitable for use as a live, attenuated vaccine in a poultry species. The method involves incubating wild-type microorganisms with a first population of heterophils taken from a member of a poultry species to generate a sample including heterophil-internalized microorganisms as well as extracellular microorganisms. A substantially pure clone of the heterophil-internalized microorganisms is used for the next step. Microorganisms from the heterophil-internalized clone are incubated with a second population of heterophils to form a next passage of heterophil-internalized microorganisms and a next passage of extracellular microorganisms. Preferably the second population of heterophils is taken from the same member of the poultry species as was used in the first passage. These procedures are repeated until at least three passages, and preferably at least five passages, are completed. A substantially pure clone of the heterophil-internalized microorganisms from the last passage is isolated to obtain a heterophil-adapted strain. The heterophil-adapted strain can be, although is not necessarily, arginine hydrolase negative.
The poultry species to which this invention is applicable include, without limitation, turkeys, guinea fowl, pigeons, quail, partridge, broiler chickens, and laying hens. The species of microorganism can include members of the genus Salmonella, for example Salmonella enteritidis. 
In a preferred embodiment, isolation of a substantially pure clone of heterophil-internalized microorganism includes incubating the heterophils with an antibiotic, washing, then disrupting the heterophils to release the internalized microorganisms. The heterophils may be incubated with more than one antibiotic. Antibiotics useful in the methods of the present invention preferably include, without limitation, aminoglycosides, or aminocyclitols. Most preferable is the use of kanamycin and gentamicin. The antibiotics can be used individually, sequentially or in any combination effective for killing or inactivating the relevant extracellular microorganisms.
In another aspect, the invention features a method of vaccinating poultry by administering a preparation of a poultry heterophil-adapted strain of microorganism to poultry, preferably by an oral route of administration. In an alternative embodiment, both a poultry heterophil-adapted strain of microorganism and cholera toxin, a mucosal adjuvant, are provided to the poultry.
In a further aspect, the present invention involves a substantially pure poultry heterophil-adapted strain of microorganism. In preferred embodiments, the microorganism is a member of the genus Salmonella. Most preferably, the Salmonella genus member is Salmonella enteritidis. The heterophil-adapted strain can be arginine hydrolase negative.
In still another aspect, the present invention features a live, attenuated vaccine for poultry comprising a preparation of a substantially pure poultry heterophil-adapted strain of microorganism. In a preferred embodiment, the microorganism is a member of the genus Salmonella. More preferably, the member of the genus Salmonella is Salmonella enteritidis, for example Salmonella enteritidis strain SETK499 or strain SETK598 having ATCC Accession Number 55770 (The American Type Culture Collection; P.O. Box 1549, Manassas, Va. 20108; Deposited: Apr. 26, 1994) In an alternative embodiment, the vaccine includes a preparation of a substantially pure poultry heterophil-adapted strain of microorganism as well as cholera toxin. Preferably, the vaccine is adapted for oral administration.
In another related aspect, the invention includes a poultry member comprising a poultry heterophil-adapted strain of microorganism. In a preferred embodiment, the microorganism is a member of the genus Salmonella, for example Salmonella enteritidis. In a further related aspect, the invention includes eggs and body parts (wings, breasts, drumsticks, or other body parts sold, for example, for human consumption) derived from the immunized poultry member. Such eggs and body parts are at a lower risk for microorganism contamination than are eggs and body parts derived from non-immunized but otherwise comparable poultry members.